CN109134648A - A kind of antibody purification process - Google Patents
A kind of antibody purification process Download PDFInfo
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- CN109134648A CN109134648A CN201811076663.6A CN201811076663A CN109134648A CN 109134648 A CN109134648 A CN 109134648A CN 201811076663 A CN201811076663 A CN 201811076663A CN 109134648 A CN109134648 A CN 109134648A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
Abstract
The embodiment of the invention provides a kind of antibody purification process, the animal being immunized with M1 recombinant protein, native protein is better than to recombinant protein qualitative response degree, the monoclonal antibody that performance is stable, antigen valence is high can be generated, antibody purification is carried out using CA-AS method, there are greater advantages for antibody recovery rate, antibody activity, antibody purity etc., and this method is easy to operate, it is reusable, lower production costs.
Description
Technical field
The present embodiments relate to biopharmaceutical technologies, more particularly, to a kind of antibody purification process.
Background technique
The technique research and development of contemporary antibody drug be based on genetic engineering, cell engineering and Fermentation Engineering basis on, in
State's Hamster Qvary (CHO) cell is clear with genetic background as main mammalian cell expression host cell, has and turns over
The features such as translating post-processing ability becomes the standard industrial cell strain of protein production, is equally also suitable for antibody protein
Expression.With the development of cell suspension cultures technology, make it possible large-scale culture, and the application of serum free medium is then
So that protein purification technique is more easy, everything has all greatly accelerated the industrialization process of antibody drug, to prepare inexpensive matter
Excellent antibody drug is laid a good foundation.
It is flourishing by nearly 30 years on the basis of Koehler and Milstein since the preparation of first monoclonal antibody
Development, monoclonal antibody technology of preparing are made that huge contribution in terms of life science and medical practice, it has also become
One of modern biotechnology pillar industry.For monoclonal antibody drug because of its targeting specific, toxic side effect is low, treatment it is efficient
The clear superiorities such as property have important application in fields such as antitumor immune response disease, organ-graft refection and virus infections.Antibody
Drug manufacturing process is from the research and development cell strain building in period, medium optimization, upstream, cell culture, downstream purification preparation to the end
Any one link of this five aspects all has critically important influence to antibody drug, wherein again the most multiple with downstream purifying process
Miscellaneous cumbersome, downstream process contains multiple steps, and currently used is that affinity chromatography, ion chromatography and hydrophobic chromatography are used in series
Method.Although all using this mode at present, the variation of tiny parameter is likely to cause huge in each technique
Variation.Downstream chromatographs will also can to upstream other than the impurity such as HCP to be removed, residual DNA and dimer polymer in technique
The virus that can be introduced is removed.
In antibody drug production process, affinity chromatography is commonly used in the acquisition phase of antibody, i.e. first purifying process step
Suddenly, antibody needed for Selective Separation goes out from fermented feed liquid, then with ion-exchange chromatography and/or hydrophobic chromatography and/or mixing
Mode chromatography, carrys out the antibody that further consummate affinity chromatography elutes.For general technology, what affinity chromatography afforded
Sample usually will do it low pH virus incubation, and can all handle the low pH sample being incubated for before chromatographing in next step,
To reach the operating condition that different chromatography methods need.
The processing of sample before traditional anion chromatography, typically a step pH, i.e., the sample being directly incubated for low pH
PH range needed for adjusting back anion chromatography loading.But to may result in antibody unstable for this method, generate precipitating to
The loss of sample is caused, it is in addition undesirable to the impurity removal effect of antibody, increase the amount of anion chromatography adsorbing contaminant, to layer
Analysis filler damages, and shortens its service life.It is miscellaneous that anion-exchange chromatography mainly removes bacterial endotoxin, host protein and DNA
Matter etc., conventional method are using flowing through (flow-through) mode, and antibody directly passes through pillar, and pollutant be absorbed it is anti-
The presence of impurity will lead to drug effect reduction in body drug, serious to will lead to human body death.Such as: containing endotoxin or by endogenous toxic material
The drug of element pollution can cause a variety of allergy after entering human body, such as: high fever, shock, even death.Many antibody, including
IgG and IgM, will form aggregation, and the presence of aggregation can generate the drug effect different with major product, will cause drug resistance antibody
Formation.Remaining host cell proteins are possible to stimulation body and the corresponding antibody of immune response generation occur, equally can be to people
Body damages.
Summary of the invention
It is anti-that the embodiment of the invention provides a kind of one kind for overcoming the above problem or at least being partially solved the above problem
Body purification process.
The embodiment of the invention provides a kind of antibody purification process, comprising:
Step 1: being inserted into expression vector pet32a in M1 genes of SEQ NO.2, M1 recombinant protein is obtained, with M1 weight
Histone is antigen, animal is immunized, then carry out cell fusion and subclone；By the cell expansion culture after above-mentioned subclone and infuse
It is incident upon mouse peritoneal, the intracorporal ascites of mouse is extracted after 7-10 days, ascites is saved at -20 DEG C；
It thaws overnight Step 2: ascites is placed in 4 DEG C, after taking-up under the conditions of 4 DEG C, 10000 × g is centrifuged 10min, takes
Clearly, 4 DEG C of preservations；Ascites is taken, dilutes 3 times with 4.4 acetate buffer solution of 0.06mol/LpH, adjusts pH to 4.8 with 1mol/L HCl；
The ratio that ascites adds 11 μ l octanoic acids is diluted in every milliliter, is stirred at room temperature down and octanoic acid is added dropwise, in adding in 30min, 4 DEG C are stood
2h；After taking-up, under the conditions of 4 DEG C, 15000 × g is centrifuged 30min, takes supernatant, and the 0.01mol/LPBS of 1/10 volume is added, and uses
1mol/LNaOH tune pH to 7.2 operates in ice face and saturated ammonium sulfate is added to 45% saturation degree, stands 30min；After taking-up,
10000 × g is centrifuged 30min, abandons supernatant, will precipitate 4 DEG C of preservations；
Step 3: it is molten with 0.01mol/LpH 7.2PB solution A weight that the precipitating in step 2 is formed sediment, through Sepharose G-
After 25 desalinations, DEAE anion chromatography column is crossed；Using the 0.01mol/LpH 7.2PB containing 0.5mol/LNaCl as eluent B, A, B
Liquid is mixed into row linear gradient elution, collects protein peak；Purification sample is stored in -20 DEG C of environment.
Further, further includes:
Step 4: detecting purified antibodies potency using indirect elisa method.
Further, in step 1, animal is immunized at least booster shots 3 times after initial immunity.
Further, the carrier pet32a is SEQ NO.3.
Further, in step 1, after cell fusion further include:
It draws the hole cell conditioned medium 100ul/ and carries out indirect ELISA detection；According to ELISA as a result, judging positive hole；Use single track
Pipettor chooses the positive hole that inspection whole plate detects, carries out second and rechecks, further confirms that positive hole.
Further, in step one kind, subcloning procedures include:
Two-wheeled subclone is done to the positive hole cell of secondary screening, is subcloned cell in limiting dilution positive hole for the first time, at most
In a hole, add HT DMEM culture medium culture, observed under the microscope after 7 days, indirect ELISA detects the hole for having clonal growth, takes
The high hole of OD value is positive hole；The cell in picking positive hole carries out second and is subcloned, and obtains positive cell strain as final system
Standby monoclonal antibody cell, and expand culture.
A kind of antibody purification process provided in an embodiment of the present invention, the animal being immunized with M1 recombinant protein, to recombinant protein
Qualitative response degree is better than native protein, can generate the monoclonal antibody that performance is stable, antigen valence is high, carries out antibody using CA-AS method
Purifying, there are greater advantages for antibody recovery rate, antibody activity, antibody purity etc., and this method is easy to operate, repeatable to make
With lower production costs.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention,
Technical solution in the embodiment of the present invention is explicitly described, it is clear that described embodiment is that a part of the invention is real
Example is applied, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creation
Property labour under the premise of every other embodiment obtained, shall fall within the protection scope of the present invention.
Influenza virus is a kind of important seasonal communicable disease, and historical produce shockingly several times issues human health and warp
Ji causes huge harm and loss.Since influenza virus is found, people are directed to the diagnosis of influenza virus and prevent
Control.Influenza A virus has the characteristics that height variation, hypotype are numerous, and certain difficulty is brought to its prevention and control.Quickly, it accurately examines
Cutout sense can provide the time for correct treatment and prevention and control influenza.The colloid gold test paper whether Accurate Diagnosis influenza virus infects
Item, and the antigen ELISA kit of detection A type stream virus, all be unable to do without the influenza monoclonal antibody that performance is stable, antigen valence is high,
Domestic biopharmaceutical company is exactly that cannot produce handy influenza virus diagnostic reagent because of not good monoclonal antibody.
M1 is made of 252 amino acid residues, molecular weight about 26KDa, it is the major structural protein of virus, accounts for influenza disease
The 30%~40% of toxalbumin total amount.The albumen has type specificity, and antigenic specificity is influenza virus parting according to it
One；Monoclonal antibody be widely used in immunology, pharmacology, cell biology, microbiology, clinical disease prevention, examine
Disconnected and treatment and the detection etc. of the violated limitation drug of livestock products.Its production method, which mainly has, induces ascites and hybridization in Mice Body
Oncocyte in vitro culture；The former production cost is low, is the main production method currently used for external application.But no matter which kind of mode
The monoclonal antibody of production, contain a large amount of foreign protein, mainly include albumin, transferrins, seralbumin, α-macroglobulin,
Lipopoteins and other host proteins etc. are not directly applicable external.Therefore subsequent purifying need to be carried out to monoclonal antibody, to reach
To requirement.But current still none of these methods is able to satisfy different purpose needs, especially anti-in prepare with scale monoclonal
Seem more prominent in body problem.
In view of the above-mentioned drawbacks in the prior art, the embodiment of the invention provides a kind of antibody purification process, comprising:
Step 1: being inserted into expression vector pet32a in M1 genes of SEQ NO.2, M1 recombinant protein is obtained, with M1 weight
Histone is antigen, animal is immunized, then carry out cell fusion and subclone；By the cell expansion culture after above-mentioned subclone and infuse
It is incident upon mouse peritoneal, the intracorporal ascites of mouse is extracted after 7-10 days, ascites is saved at -20 DEG C；
It thaws overnight Step 2: ascites is placed in 4 DEG C, after taking-up under the conditions of 4 DEG C, 10000 × g is centrifuged 10min, takes
Clearly, 4 DEG C of preservations；Ascites is taken, dilutes 3 times with 4.4 acetate buffer solution of 0.06mol/LpH, adjusts pH to 4.8 with 1mol/L HCl；
The ratio that ascites adds 11 μ l octanoic acids is diluted in every milliliter, is stirred at room temperature down and octanoic acid is added dropwise, in adding in 30min, 4 DEG C are stood
2h；After taking-up, under the conditions of 4 DEG C, 15000 × g is centrifuged 30min, takes supernatant, and the 0.01mol/LPBS of 1/10 volume is added, and uses
1mol/LNaOH tune pH to 7.2 operates in ice face and saturated ammonium sulfate is added to 45% saturation degree, stands 30min；After taking-up,
10000 × g is centrifuged 30min, abandons supernatant, will precipitate 4 DEG C of preservations；
Step 3: it is molten with 0.01mol/LpH 7.2PB solution A weight that the precipitating in step 2 is formed sediment, through Sepharose G-
After 25 desalinations, DEAE anion chromatography column is crossed；Using the 0.01mol/LpH 7.2PB containing 0.5mol/LNaCl as eluent B, A, B
Liquid is mixed into row linear gradient elution, collects protein peak；Purification sample is stored in -20 DEG C of environment.
Further, further includes:
Step 4: detecting purified antibodies potency using indirect elisa method.
Further, in step 1, animal is immunized at least booster shots 3 times after initial immunity.
Further, the carrier pet32a is SEQ NO.3.
Further, in step 1, after cell fusion further include:
It draws the hole cell conditioned medium 100ul/ and carries out indirect ELISA detection；According to ELISA as a result, judging positive hole；Use single track
Pipettor chooses the positive hole that inspection whole plate detects, carries out second and rechecks, further confirms that positive hole.
Further, in step one kind, subcloning procedures include:
Two-wheeled subclone is done to the positive hole cell of secondary screening, is subcloned cell in limiting dilution positive hole for the first time, at most
In a hole, add HT DMEM culture medium culture, observed under the microscope after 7 days, indirect ELISA detects the hole for having clonal growth, takes
The high hole of OD value is positive hole；The cell in picking positive hole carries out second and is subcloned, and obtains positive cell strain as final system
Standby monoclonal antibody cell, and expand culture.
A kind of antibody purification process provided in an embodiment of the present invention, the animal being immunized with M1 recombinant protein, to recombinant protein
Qualitative response degree is better than native protein, can generate the monoclonal antibody that performance is stable, antigen valence is high, carries out antibody using CA-AS method
Purifying, there are greater advantages for antibody recovery rate, antibody activity, antibody purity etc., and this method is easy to operate, repeatable to make
With lower production costs.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features；
And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and
Range.
Claims (6)
1. a kind of antibody purification process characterized by comprising
Step 1: being inserted into expression vector pet32a in M1 genes of SEQ NO.2, M1 recombinant protein is obtained, egg is recombinated with the M1
White is antigen, animal is immunized, then carry out cell fusion and subclone；By the cell expansion culture after above-mentioned subclone and it is injected to
Mouse peritoneal extracts the intracorporal ascites of mouse, ascites is saved at -20 DEG C after 7-10 days；
It thaws overnight Step 2: ascites is placed in 4 DEG C, after taking-up under the conditions of 4 DEG C, 10000 × g is centrifuged 10min, take supernatant, 4 DEG C
It saves；Ascites is taken, dilutes 3 times with 0.06mol/LpH4.4 acetate buffer solution, adjusts pH to 4.8 with 1mol/L HCl；By every milli
It rises dilution ascites and adds the ratios of 11 μ l octanoic acids, be stirred at room temperature down and octanoic acid is added dropwise, in being added in 30min, 4 DEG C of standing 2h；It takes
After out, under the conditions of 4 DEG C, 15000 × g is centrifuged 30min, takes supernatant, and the 0.01mol/LPBS of 1/10 volume is added, uses 1mol/
LNaOH tune pH to 7.2 operates in ice face and saturated ammonium sulfate is added to 45% saturation degree, stands 30min；After taking-up, 10000 × g
It is centrifuged 30min, abandons supernatant, 4 DEG C of preservations will be precipitated；
Step 3: it is molten with 0.01mol/L pH 7.2PB solution A weight that the precipitating in step 2 is formed sediment, removed through Sepharose G-25
After salt, DEAE anion chromatography column is crossed；Using the 0.01mol/L pH 7.2PB of the NaCl containing 0.5mol/L as eluent B, A, B liquid
It is mixed into row linear gradient elution, collects protein peak；Purification sample is stored in -20 DEG C of environment.
2. antibody purification process described according to claim 1, which is characterized in that further include:
Step 4: detecting purified antibodies potency using indirect elisa method.
3. antibody purification process described according to claim 1, which is characterized in that in step 1, immune animal is exempted from for the first time
At least booster shots 3 times after epidemic disease.
4. antibody purification process described according to claim 1, which is characterized in that the carrier pet32a is SEQ NO.3.
5. antibody purification process described according to claim 1, which is characterized in that in step 1, after cell fusion further include:
It draws the hole cell conditioned medium 100ul/ and carries out indirect ELISA detection；According to ELISA as a result, judging positive hole；With single track liquid relief
Device chooses the positive hole that inspection whole plate detects, carries out second and rechecks, further confirms that positive hole.
6. the antibody purification process according to claim 5, which is characterized in that in step one kind, subcloning procedures include:
Two-wheeled subclone is done to the positive hole cell of secondary screening, cell in limiting dilution positive hole is subcloned for the first time, until multiple holes
In, add HT DMEM culture medium culture, observed under the microscope after 7 days, indirect ELISA detects the hole for having clonal growth, takes OD value
High hole is positive hole；The cell in picking positive hole carries out second and is subcloned, and obtains positive cell strain as finally preparing
Monoclonal antibody cell, and expand culture.
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|---|---|---|---|
| CN201811076663.6A CN109134648A (en) | 2018-09-14 | 2018-09-14 | A kind of antibody purification process |
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| CN201811076663.6A CN109134648A (en) | 2018-09-14 | 2018-09-14 | A kind of antibody purification process |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113861289A (en) * | 2021-10-18 | 2021-12-31 | 南京京达生物技术有限公司 | Method for purifying goat anti-human IgM antibody |
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